Biosensor

ABSTRACT

A biosensor can supply a sample solution accurately and easily, and includes a capillary for collecting a sample solution and analyzes a specific substance in the sample solution, an air hole, and at least two supply ports, i.e., a sample supply port and an auxiliary sample supply port, so that supply of the sample solution can be performed from either of the supply ports. Even when the sample supply port is closed up with a fingertip or the like and supply of the sample solution is stopped, the sample solution can be quickly supplied from the other auxiliary sample supply port.

TECHNICAL FIELD

The present invention relates to a biosensor for analyzing a specificcomponent in a sample solution, and more particularly, to a biosensorwhich collects a small amount of sample solution by capillary phenomenononto a small-size test specimen, and analyzes the sample solution.

BACKGROUND ART

A biosensor is a sensor for determining a quantity of a base substancein a sample solution, which utilizes a molecule recognizing ability of abiological material such as micro-organism, enzyme, antibody, DNA, RNAor the like to employ the biological material as a moleculediscrimination element. To be specific, the biosensor determines aquantity of a base substance contained in a sample solution by utilizinga reaction which occurs when a biological material recognizes anobjective substrate, such as consumption of oxygen due to respiration ofa micro-organism, enzyme reaction, light emission, and the like. Amongvarious kinds of biosensors, an enzyme sensor has come into practicaluse. For example, an enzyme sensor as a biosensor for glucose, lacticacid, cholesterol, or amino acid has been utilized for medical analysisand food industry. In this enzyme sensor, an electron carrier is reducedby electrons that are generated due to a reaction between a basesubstance included in a sample solution as an analyte and enzyme or thelike, and a measurement unit electrochemically measures a reductionquantity of the electron carrier, thereby performing quantitativeanalysis for the sample.

There have been proposed various types of biosensors. For example, as abiosensor that facilitates measurement of a blood glucose level, thereis a biosensor comprising a first insulating substrate on which a pairof electrodes and a reagent layer are formed, a second insulatingsubstrate bonded to the first insulating substrate via a spacer, and acapillary for collecting a sample solution, which is provided betweenthe both insulating substrates. The biosensor is constituted such thatblood obtained by puncturing the human body is introduced by capillaryphenomenon into the capillary from a sample supply port that opens atone ends of the both substrates.

In this biosensor, however, there is a possibility that the blood is notsuccessfully introduced into the capillary depending on the angle of thebiosensor when the blood is applied onto the sample supply port, andthereby the blood might be attached to the outer surface of theinsulating substrate by mistake. In this case, even when the user triesto supply the blood again, the blood attached to the outer surfaceimpedes the user from successfully supplying the blood into thecapillary, resulting in faulty measurement and measurement errors.

In order to solve this problem, the inventors of the present inventionhave proposed a biosensor in which the ends of the both substrates whichconstitute the sample supply port are formed in different shapes whenviewed planarly so that blood can always be introduced into thecapillary successfully without being influenced by the angle of thebiosensor when the blood is applied (refer to Patent Document 1).

FIG. 8 illustrates an exploded perspective view and a cross-sectionalview of the biosensor disclosed in Patent Document 1. In FIG. 8,reference numeral 1 denotes a first insulating substrate, and ameasurement electrode 2, a counter electrode 3, and a detector electrode4, which comprise an electric conducting material, are formed on thefirst insulating substrate 1.

The conventional biosensor 800 is formed by bonding the first insulatingsubstrate 1, a spacer 6, and a second insulating substrate 8 together,and a capillary 7 is formed by the existence of a notch in the spacer 6.A test sample is introduced into the capillary 7 from its front end by asample supply port 13 that is formed by the bonding and an air hole 9formed through the insulating substrate 1.

Further, the measurement electrode 2, the counter electrode 3, and thedetector electrode 4 which are formed on the first insulating substrate1 are exposed in the capillary 7, and a reagent layer 5 is formed in aposition opposed to these electrodes.

A measurement instrument (not shown) having terminals to be connected toleads 10, 11, and 12 of the electrodes is inserted in the biosensorbefore introduction of blood, and variation in the electriccharacteristics which occurs due to a reaction of the blood with thereagent is detected between the measurement electrode 2 and the counterelectrode 3 after introduction of blood, thereby measuring a glucoseconcentration.

Patent Document 1: Japanese Published Patent Application No.2002-168821).

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

In blood glucose measurement in recent years, it is desired to minimizethe quantity of blood to be collected in order to reduce pain of adiabetic patient as much as possible. Therefore, development of abiosensor in which the size of the capillary for collecting blood andthe size of the sample supply port are further reduced has beenprogressed.

However, such miniaturization in the conventional biosensor has caused aproblem that the sample supply port is easily closed up when adeformable object such as a finger chip is pressed thereto.

FIG. 9 shows a state where blood is aspirated in the conventionalbiosensor.

As shown in FIG. 9( a), when the sample supply port 13 is closed up by afingertip, supply of blood is interrupted, and the blood is notcompletely filled in the capillary 7 but stops in the middle of thecapillary 7. Then, shortage of sample quantity occurs, which may preventmeasurement, or display of incorrect results. Further, even when thecapillary is completely filled with the blood by rightly separating thefinger as shown in FIG. 9( b) after the finger has once closed thesample supply port 13, there occurs a difference in dissolution of thereagent layer due to the initially introduced blood, resulting invariations in measurement, and therefore, accurate measurement cannot becarried out.

Although it might be considered that the difference in the shapesbetween the first insulating substrate and the second insulatingsubstrate may be further increased to prevent the fingertip from closingthe sample supply port, this is a distant idea. The reason is asfollows. If the difference in the shapes is increased too much, not onlythe blood stored inside the capillary but also the blood stored outsidethe capillary increases, and thus more blood is required.

The present invention is made to solve the above-described problems andhas for its object to provide a biosensor having a construction that canreliably collect a sample solution into a capillary even when thequantity of the sample solution is very small.

Measures to Solve the Problems

In order to solve the above-mentioned problems, there is provided abiosensor which is formed by bonding a first insulating substrate and asecond insulating substrate together, and comprises a sample supply portwhich opens at one end of the both substrates, to which a samplesolution is applied, a capillary communicated with the sample supplyport, into which the applied sample solution is introduced by capillaryphenomenon, and an air hole which is positioned at an end of thecapillary and communicated with air inside the capillary, the supplyport, the capillary, and the air hole being formed by the bonding of theboth insulating substrates, wherein at least one auxiliary sample supplyport communicated with the capillary, through which the applied samplesolution is introduced into the capillary, is provided in the vicinityof the sample supply port.

Further, the auxiliary sample supply port is obtained by forming athrough-hole in the first insulating substrate or the second insulatingsubstrate so as to leave a portion of the insulating substrate betweenthe auxiliary sample supply portion and the sample supply port.

Further, a spacer having a groove to provide the sample supply port, theauxiliary sample supply port, and the capillary is disposed between thefirst insulating substrate and the second insulating substrate, and theauxiliary sample supply port is provided at the ends of the bothsubstrates.

EFFECTS OF THE INVENTION

According to the present invention, since a biosensor which has acapillary structure and performs measurement with a very small quantityof sample is constituted as described above, even when a sample supplyport is closed up by elastic skin such as fingertip, brachial region, orabdominal region of a test subject, it is possible to perform reliableaspiration of the sample solution from an auxiliary sample supply portinto the capillary.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an exploded perspective view and a cross-sectionalview of a biosensor 100 according to a first embodiment of the presentinvention.

FIG. 2 illustrates an exploded perspective view and a cross-sectionalview of a biosensor 200 according to a modification of the firstembodiment.

FIG. 3 illustrates an exploded perspective view and a cross-sectionalview of a biosensor 300 according to another modification of the firstembodiment.

FIG. 4 illustrates an exploded perspective view and a cross-sectionalview of a biosensor 400 according to still another modification of thefirst embodiment.

FIG. 5 illustrates an exploded perspective view and a cross-sectionalview of a biosensor 500 according to a second embodiment of the presentinvention.

FIG. 6 illustrates an exploded perspective view and a cross-sectionalview of a biosensor 600 according to a third embodiment of the presentinvention.

FIG. 7 illustrates an exploded perspective view and a cross-sectionalview of a biosensor 700 according to a comparison example of the presentinvention.

FIG. 8 illustrates an exploded perspective view and a cross-sectionalview of the conventional biosensor 800.

FIG. 9 is a cross-sectional view illustrating a state where blood isaspirated in the conventional biosensor 800.

FIG. 10 is a cross-sectional view illustrating a state where blood isaspirated in the biosensor 100 according to the first embodiment.

DESCRIPTION OF THE REFERENCE NUMERALS

-   -   100 . . . biosensor    -   200 . . . biosensor    -   300 . . . biosensor    -   400 . . . biosensor    -   500 . . . biosensor    -   600 . . . biosensor    -   700 . . . biosensor    -   800 . . . biosensor    -   1 . . . first insulating substrate    -   2 . . . measurement electrode    -   3 . . . counter electrode    -   4 . . . detector electrode    -   5 . . . reagent layer    -   6 . . . spacer    -   7 . . . capillary    -   8 . . . second insulating substrate    -   9 . . . air hole    -   10 . . . lead    -   11 . . . lead    -   12 . . . lead    -   13 . . . sample supply port    -   14 . . . auxiliary sample supply port    -   15 . . . notch    -   16 . . . blood    -   17 . . . fingertip

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, embodiments of a biosensor according to the presentinvention will be described taking a blood glucose sensor as an examplewith reference to the drawings.

Embodiment 1

FIG. 1 illustrates an exploded perspective view and a cross-sectionalview of a biosensor 100 according to a first embodiment of the presentinvention.

In the biosensor 100 shown in FIG. 1, reference numeral 1 denotes afirst insulating substrate having a portion near a front end beingformed approximately in a semicircular shape, and a portion that followsthe front end to reach a rear end being formed in a rectangle. Ameasurement element 2, a counter electrode 3, and a detector electrode 4which are composed of an electric conducting material are formed on thefirst insulating substrate 1. Reference numeral 8 denotes a secondinsulating substrate which is formed in a shape similar to that of thefirst insulating substrate 1, reference numeral 6 denotes a spacer whichis disposed between the first insulating substrate 1 and the secondinsulating substrate 8 and is formed in a shape similar to those of theboth insulating substrates, and reference numeral 7 denotes a capillarywhich is formed so as to form an approximately rectangle convex portionin the vicinity of the front end of the spacer, along the longitudinaldirection of the spacer.

The biosensor 100 is formed by bonding the first insulating substrate 1,the spacer 6, and the second insulating substrate 8 together, and thecapillary 7 is formed by existence of the above-mentioned notch in thespacer 6. A test sample is introduced into the capillary 7 by a samplesupply port 13 that is formed by the bonding, and an air hole 9 that isprovided through the first insulating substrate 1 in a position opposedto a rear end of the capillary 7.

Further, reference numerals 10, 11, and 12 denote leads of themeasurement electrode 2, the counter electrode 3, and the detectorelectrode 4, respectively, which correspond to the rear end portions ofthe respective electrodes disposed on the first insulating substrate 1,and reference numeral 13 denotes a sample supply port which is formed bythat a forward space portion of the capillary 7 is sandwiched by thefirst and second insulating substrates 1 and 8.

Further, the measurement electrode 2, the counter electrode 3, and thedetector electrode 4 formed on the first insulating substrate 1 areexposed in the capillary 7, and a reagent layer 5 is disposed in aposition opposed to these electrodes.

When performing measurement using the biosensor 100 of the firstembodiment, variations in the electric characteristics between themeasurement electrode 2 and the counter electrode 3 are detected withthe biosensor 100 being inserted in a measurement instrument (not shown)having terminals which are to be connected to the leads 10, 11, and 12of the respective electrodes 2, 3, and 4, thereby to analyze thecharacteristics of the test sample.

While the detector electrode 4 functions as an electrode for detecting ashortage in the quantity of the sample, it may be used as a referenceelectrode or a portion of the counter electrode.

While in FIG. 1 the respective electrodes 2, 3, and 4 are disposed onthe first insulating substrate 1, these electrodes may be partiallydisposed on the opposed second insulating substrate 8 as well as on thefirst insulating substrate 1.

Preferable materials of the first insulating substrate 1, the spacer 6,and the second insulating substrate 8 include polyethyleneterephthalate, polycarbonate, and polyimide. The thicknesses of thefirst and second insulating substrates are desired to be 0.1 to 5.0 mm.

Further, the electric conducting material constituting the respectiveelectrodes 2, 3, and 4 may include a single substance such as a noblemetal (gold, platinum, or palladium) or carbon, or a complex substratesuch as carbon paste or a noble metal paste. Sputtering or the like isadopted for the former substance while screen printing or the like isadopted for the latter substance, thereby easily forming the electricconducting layer on the first insulating substrate 1 or the secondinsulating substrate 8.

Further, when forming the respective electrodes, initially an electricconducting layer is formed on the entire surface or a portion of thefirst insulating substrate 1 or the second insulating substrate 8 by theabove-mentioned sputtering or screen printing, and then slits are formedin the electric conducting layer using a laser or the like, therebyfabricating the separated electrodes. Alternatively, the respectiveelectrodes can be similarly produced by screen printing or sputteringusing a print board or a mask board on which electrode patterns havealready been formed.

The reagent layer 5 including enzyme, electron carrier, hydrophilicmacromolecule, and the like is formed on the electrodes 2, 3, and 4. Theenzyme may be any of glucose oxidase, lactate oxidase, cholesteroloxidase, cholesterol esterase, uricase, ascorbate oxidase, bilirubinoxidase, glucose dehydrogenase, and lactate dehydrogenase. The electroncarrier may be any of potassium ferricyanide, p-benzoquinone and itsderivative, phenazine methosulfate, methylene blue, and ferrocene andits derivative.

The hydrophilic macromolecule may be any of carboxymethyl cellulose,hydroxyethyl cellulose, hydroxypropyl cellulose, methyl cellulose, ethylcellulose, ethylhydroxyethyl cellulose, carboxymethylethyl cellulose,polyvinyl alcohol, polyvinylpyrrolidone, polyamino acid such aspolylysine, polystyrene sulfonate, gelatine and its derivative, acrylicacid and its salt, and agarose gel and its derivative.

Next, the capillary 7 to which blood is to be supplied is formed bybonding the first insulating substrate 1 and the second insulatingsubstrate 8 with the spacer 6 between them. The sample supply port 13through which the blood is to be introduced into the capillary 7 isopened at the ends of the first insulating substrate 1 and the secondinsulating substrate 8.

In this first embodiment, the thickness of the spacer 6 is 0.025 to 0.5mm, the width of the capillary 7 is 0.1 to 10 mm, and the volume of thecapillary 7 is 0.1 to 5 μL.

The construction of the first embodiment is characterized by that anauxiliary sample supply port 14 penetrating through the secondinsulating substrate 8 on the capillary 7 is provided. After thisauxiliary sample supply port 14 is formed through the second insulatingsubstrate 8, the second insulating substrate 8 is bonded to the firstinsulating substrate 1 and the spacer 6, thereby completing thebiosensor.

Since the auxiliary sample supply port 14 is provided, even when thesample supply port 13 is closed up with a finger chip when applying theblood and thereby supply of the blood from the sample supply port 13 isblocked, the blood can be introduced into the capillary from theauxiliary sample supply port 14 provided through the second insulatingsubstrate 8 as shown in FIG. 10, whereby the capillary 7 can becompletely filled with the blood.

This auxiliary sample supply port 14 is desired to be provided in aposition to which the sample solution is always attached when the samplesolution is supplied. Hereinafter, a description will be given of theposition, size, shape, and number of the auxiliary sample supply port14.

The distance between the sample supply port 13 and the auxiliary samplesupply port 14, i.e., the size of A shown in the cross-sectional view ofFIG. 1( b), is desirably at least 0.05 to 5.0 mm. When the distance issmaller than 0.05 mm, there is a possibility that the two supply portsmight be connected and the effect as the auxiliary sample supply port isreduced. Further, in the recent biosensor which is desired to minimizethe quantity of blood, if the distance is larger than 5.0 mm, it becomesdifficult to apply the sample to the sample supply port 13 and to theauxiliary sample supply port 14 simultaneously.

The area of the auxiliary sample supply port 14 is desired to be 0.01 to3.0 mm². When the area is smaller than 0.01 mm², the auxiliary samplesupply port 14 lacks the ability of aspirating the sample solution, andthereby the supply speed is reduced or the supply is stopped halfway.When the area is larger than 3.0 mm², the size of the capillary must beincreased, which leads to an increase in the quantity of the sample, andtherefore, this is a distant idea.

It is desired to process the auxiliary sample supply port 14 using alaser. Although press cutting, die cutting, and Thomson cutting are alsoapplicable for processing the supply port, laser processing is mostpreferable because it enables microfabrication.

A plurality of auxiliary sample supply ports 14 may be provided on thesecond insulating substrate 8, with favorable effects. Further, theshape of the auxiliary sample supply port 14 is not restricted to thatmentioned above so long as the above-mentioned conditions are satisfied.For example, it may be circular, oval, linear, rectangular, triangular,or the like.

Further, while the auxiliary sample supply port 14 is provided on thesecond insulating substrate 8, it may be provided on the firstinsulating substrate 1. At this time, the position, shape, and size ofthe auxiliary sample supply port 14 are identical to those mentionedabove.

Further, the shape of the biosensor 100 is not restricted to that of thefirst embodiment shown in FIG. 1, and the same effects as mentionedabove can be achieved even when the biosensor has a shape according to amodification shown in FIG. 2 or a shape according to anothermodification shown in FIG. 3.

To be specific, a biosensor 200 according to a modification of the firstembodiment shown in FIG. 2 has plural auxiliary sample supply ports 14 aand 14 b.

Further, a biosensor 300 according to another modification of the firstembodiment shown in FIG. 3 has a rectangular auxiliary sample supplyport 14.

Furthermore, FIG. 4 shows a biosensor 400 according to still anothermodification of the first embodiment. This biosensor 400 is constitutedsuch that the first insulating substrate 1 and the second insulatingsubstrate 8 which form the capillary 7 are bonded together shifted fromeach other so that the end portions thereof viewed planarly are locatedin different positions.

That is, in FIG. 4, the second insulating substrate 8 and the spacer 6are protruded by 0.1 to 1.0 mm toward the sample supply port 13 withrespect to the first insulating substrate 1.

The biosensors 200, 300, and 400 shown in FIGS. 2, 3, and 4 also achievethe same effects as the biosensor 100 shown in FIG. 1.

When the electrodes 2, 3, and 4 and the reagent layer 5 forelectrochemically analyzing a specific substance in the sample solutionare provided inside the capillary 7, it is desired that these electrodes2, 3, 4 and the reagent layer 5 are not disposed at a position on thefirst insulating substrate 1 directly beneath the auxiliary samplesupply port 14.

If the auxiliary sample supply port 14 is disposed above the electrodes2, 3, and 4, the sample solution on the electrodes is likely to vary,and this variation may cause undesirable variation in the responsevalue.

The biosensors 200, 300, and 400 shown in FIGS. 2, 3, and 4 also achievethe same effects as the biosensor 100 shown in FIG. 1.

Further, in the above-mentioned biosensors 100, 200, 300, and 400, it isdesired that a surface-activating treatment is applied to the entiretyor a portion of the inner wall of the capillary 7. Thereby, even whenthe area of the sample supply port is small, the capillary can speedilyaspirate the sample solution.

Further, it is desired that a surface-activating treatment is applied tothe inner side of the auxiliary sample supply port 14, or the entireinner wall of the capillary, or a portion of the inner wall of thecapillary in the vicinity of the auxiliary sample supply port.

When a surface-activating treatment is applied to the inner side of theauxiliary sample supply port 14 or the inner wall of the capillary,aspiration of the sample solution is quickly started as soon as thesample solution contacts the auxiliary sample supply port 14, andthereby the capillary is filled with the sample solution before thesupply port is closed up by a fingertip or the like.

The surface-activating treatment includes coating of a nonionic,cationic, anionic, or zwitterionic surfactant, corona dischargetreatment, and physical processing to form fine concavities andconvexities on the surface.

As described above, according to the biosensor of the first embodiment,even when the sample supply port 13 is closed up while the samplesolution is being supplied, the sample solution is speedily suppliedfrom the auxiliary sample supply port 14, and thereby the samplesolution is aspirated into the capillary accurately and easily.

Embodiment 2

FIG. 5 illustrates an exploded perspective view and a cross-sectionalview of a biosensor 500 according to a second embodiment of the presentinvention.

In the biosensor 500 of the second embodiment shown in FIG. 5, auxiliarysample supply ports 14 are provided on both the first insulatingsubstrate 1 and the second insulating substrate 8.

Since the auxiliary sample supply ports 14 are provided on the twoinsulating substrates 1 and 8, respectively, even if the sample isapplied from a biased angle, the sample can be reliably aspirated intothe space 6.

Further, as described in the first embodiment, plural auxiliary samplesupply ports 14 may be provided on the respective substrates with thesame effects as mentioned above.

Further, the shape of the auxiliary sample supply port 14 is notparticularly restricted, and it may be circular, oval, linear,rectangular, or triangular.

Embodiment 3

FIG. 6 illustrates an exploded perspective view and a cross-sectionalview of a biosensor 600 according to a third embodiment of the presentinvention.

In the biosensor 600 of the third embodiment shown in FIG. 6, thecapillary 7 branches in a Y shape in the vicinity of the front end, andone of the branches serves as the sample supply port 13 while the otherserves as the auxiliary sample supply port 14.

In this third embodiment, since the spacer 6 is provided with the twosample supply ports, the same effects as those of the first and secondembodiments are achieved. Further, since the sample supply port 13 andthe auxiliary sample supply port 14 can be simultaneously patterned inthe spacer 6, the number of process steps in the sensor fabrication canbe reduced.

Hereinafter, a specific example of the present invention will bedescribed in detail.

A biosensor constituted as mentioned below is used as an example.

After a palladium thin film having a thickness of about 8 nm is formedby sputtering over the entire surface of a first insulating substratecomprising polyethylene terephthalate, slits are partially formed in thethin film by using a YAG laser, thereby separately forming a measurementelectrode, a counter electrode, and a detector electrode.

Thereafter, an aqueous solution containing glucose dehydrogenase as anenzyme and potassium ferricyanide as an electron carrier is droppedcircularly so as to partially cover the counter electrode and thedetector electrode with the measurement electrode being in the center,and then dried, thereby forming a reagent layer. Further, a spacercomprising polyethylene terephthalate and a second insulating substratealso comprising polyethylene terephthalate are bonded onto the firstinsulating substrate.

A surface-activating treatment is previously applied to the surface ofthe second insulating substrate on the sample supply port side, and anair hole is formed through the second insulating substrate, and further,an auxiliary sample supply port is formed at a position apart by 0.2 mmfrom the sample supply port.

The above-mentioned members are bonded together to complete a biosensorhaving a capillary into which blood is introduced, which has the sameconstruction as that shown in FIG. 1.

In order to confirm the effects of the present invention, there arefabricated fourteen types of sensors as follows:

a conventional biosensor 800 shown in FIG. 8 ((1));

biosensors 100 according to the first embodiment shown in FIG. 1,wherein the aperture areas of the auxiliary sample supply ports 14 are0.005 mm², 0.010 mm², 0.030 mm², and 0.100 mm², respectively ((2), (3),(4), (5));

biosensors 200 according to a modification of the first embodiment shownin FIG. 2, wherein the number of the auxiliary sample supply ports 14 istwo (area: 0.003 mm²), two (area: 0.050 mm²), four (area: 0.01 mm²), andnine (area: 0.01 mm²), respectively ((6), (7), (8), (9));

a biosensor 300 according to another modification of the firstembodiment shown in FIG. 3, wherein the auxiliary sample supply port 14is rectangle in shape ((10));

a biosensor 500 according to the second embodiment shown in FIG. 5,wherein the auxiliary sample supply ports 14 are formed on both thefirst insulating substrate 1 and the second insulating substrate 8((11));

a biosensor having an auxiliary sample supply port on the firstinsulating substrate ((12));

a biosensor 600 according to the third embodiment shown in FIG. 6,wherein the capillary 7 is Y-shaped ((13)); and

a biosensor 700 as a sensor for comparison shown in FIG. 7, wherein agroove-shaped slit 15 is formed at a front end of a second insulatingsubstrate 8, and a sample supply port 13 and an auxiliary sample supplyport formed by the slit 15 are connected ((14)).

Then, 2 μL of blood which is sufficient to completely fill the samplesupply port of the biosensor of this example is collected on afingertip, the finger is pressed against the sample supply port, and theblood aspiration state when the sample supply port is closed up ischecked.

Table 1 shows the test results.

TABLE 1 area of number of AUX supply AUX supply result sample port port1 2 3 4 5 conventional sensor (1) — 0 X X X X X invention AUX supplyport (2) 0.005 mm² 1 Δ Δ X Δ ◯ sensor on 2nd insulating (3) 0.010 mm² 1◯ ◯ ◯ ◯ ◯ substrate (4) 0.030 mm² 1 ◯ ◯ ◯ ◯ ◯ (5) 0.100 mm² 1 ◯ ◯ ◯ ◯ ◯(6) 0.003 mm² 2 Δ Δ X Δ Δ (7)  0.05 mm² 2 ◯ ◯ ◯ ◯ ◯ (8)  0.01 mm² 4 ◯ ◯◯ ◯ ◯ (9)  0.01 mm² 9 ◯ ◯ ◯ ◯ ◯ rectangular AUX (10)   0.01 mm² 1 ◯ ◯ ◯◯ ◯ supply port (FIG. 3) AUX supply ports (11)   0.01 mm² 2 ◯ ◯ ◯ ◯ ◯ onboth insulating substrates (FIG. 5) AUX supply port (12)   0.01 mm² 1 ◯◯ ◯ ◯ ◯ on 1st insulating substrate Y-shaped (13)   0.15 mm² 1 ◯ ◯ ◯ ◯ ◯capillary (FIG. 6) comparison main supply port (14)  0.010 mm² 0 X X X ΔΔ sensor short-circuited with AUX supply port (FIG. 7) ◯: Speedily andaccurate aspiration is performed even when finger is pressed. Δ:Aspiration is lowered in speed or stopped halfway when finger ispressed. X: Aspiration is stopped when finger is pressed.

As is evident from Table 1, in the conventional biosensor having noauxiliary sample supply port, when the finger is pressed against thesample supply port, aspiration is stopped in all the results. This isbecause the sample supply port is closed up by pressing an elasticobject such as a fingertip, and thereby supply of the sample solution isprevented.

Further, when the area of the auxiliary sample supply port is 0.005 mm²,aspiration speed is lowered when the finger is pressed against thesupply port. It is estimated that the area of the auxiliary samplesupply port is small and insufficient to introduce the blood into thecapillary.

When the area of the auxiliary sample supply port is equal to or largerthan 0.01 mm², speedily aspiration is carried out even when the fingeris pressed against the supply port. It is estimated that even when thesample supply port is closed up and supply of the sample solution israte-limited, the sample solution is speedily supplied from theauxiliary sample supply port.

When plural auxiliary sample supply ports are provided, the same effectscan be obtained so long as the total of the areas of the supply ports isequal to or larger than 0.01 mm².

In the case where the main supply port and the auxiliary supply port areconnected and a groove-shaped slit is formed at the front end of thesecond insulating substrate as shown in FIG. 7, aspiration is stopped orlowered in speed when the finger is pressed against the main supplyport, even though the area of the groove is 0.01 mm². It is estimatedthat when the sample supply port and the auxiliary supply port areconnected, the finger pressed against the sample supply port undesirablyadheres tightly to the inside of the auxiliary supply port, and therebythe auxiliary supply port becomes incapable of performing its function.

On the other hand, favorable results can be obtained with respect to thebiosensor having a rectangle auxiliary sample supply port (refer to FIG.3), the biosensor having auxiliary sample supply ports on both the firstand second insulating substrates 1 and 2 (refer to FIG. 5), thebiosensor having an auxiliary sample supply port on the insulatingsubstrate 1, and the biosensor having a Y-shaped capillary (refer toFIG. 6).

When performing measurement with a very small quantity of samplesolution as in this example, if the sample supply port and the auxiliarysample supply port are separated by 5 mm or more, it is difficult tomake the sample contact these ports simultaneously, and favorableeffects cannot be obtained.

Also when the area of the auxiliary sample supply port is equal to orlarger than 3 mm², it is difficult to make the sample contact theentirety of the auxiliary supply port for the same reason as mentionedabove, and the auxiliary supply port cannot perform its function.

APPLICABILITY IN INDUSTRY

A biosensor according to the present invention is applicable to a bloodglucose sensor, a cholesterol sensor, a lactic acid sensor, an alcoholsensor, an amino acid sensor, a fructose sensor, and a sucrose sensor,which collect a very small quantity of sample solution into a capillaryand perform analysis. Further, samples used for the analysis may includeliquid samples such as blood, urine, sweat, saliva, drinkable water, andsewage water.

1-12. (canceled)
 13. A biosensor comprising a first insulating substrateand a second insulating substrate bonded together; a sample supply portwhich opens at one end of the bonded insulating substrates, and to whicha sample solution is applied; a capillary communicated with the samplesupply port, and into which the applied sample solution is introduced;an air hole which is positioned at an end of the capillary andcommunicated with air inside the capillary; and a spacer having agroove, the spacer being disposed between the first insulating substrateand the second insulating substrate, wherein said sample supply port andsaid capillary are formed by bonding the insulating substrates together,wherein an auxiliary sample supply port communicated with saidcapillary, through which the applied sample solution is introduced intothe capillary, is provided in the vicinity of said sample supply port,wherein the groove in the spacer forms the capillary and the samplesupply port, and wherein the auxiliary sample supply port is disposed atone end of the bonded insulating substrates.
 14. A biosensor as definedin claim 13, wherein a total aperture area of said auxiliary samplesupply port is 0.01 mm² to 3 mm².
 15. A biosensor as defined in claim13, wherein a distance between said sample supply port and saidauxiliary sample supply port is 0.05 to 5 mm.
 16. A biosensor as definedin claim 13, wherein said through-hole for the auxiliary sample supplyport is fabricated using a laser.
 17. A biosensor as defined in claim13, wherein a surface-activating treatment is applied to at least aportion of a surface of the first insulating substrate or the secondinsulating substrate, said portion facing the capillary.
 18. A biosensoras defined in claim 13, wherein electrodes and a reagent layer forelectrochemically analyzing a specific substance in the sample solutionare provided on a surface of the first insulating substrate or thesecond insulating substrate, said surface facing the capillary.
 19. Abiosensor as defined in claim 18, wherein the first insulating substrateand the second insulating substrate have different shapes at an end ofthe biosensor where the sample supply port is formed.
 20. A biosensor asdefined in claim 19, wherein the electrodes or the reagent layer are/isnot provided on the first or second insulating substrate which isopposed to the auxiliary sample supply port.
 21. A biosensor as definedin claim 13, wherein a surface-activating treatment is applied to aninner wall of the auxiliary sample supply port.
 22. A biosensor asdefined in claim 13, wherein said auxiliary supply port is a firstauxiliary supply port, and the biosensor comprises at least onadditional auxiliary supply port, and wherein a total aperture area ofthe auxiliary sample supply ports is 0.01 mm² to 3 mm².
 23. A biosensoras defined in claim 13, wherein said auxiliary sample supply port isdisposed between said air hole and said sample supply port.
 24. Abiosensor as defined in claim 13, wherein said auxiliary supply port isa first auxiliary supply port and the biosensor includes at least oneadditional auxiliary supply port, and wherein each of said auxiliarysample supply ports is disposed between said air hole and said samplesupply port.
 25. A biosensor as defined in claim 13, wherein saidauxiliary sample supply port and said air hole are disposed in saidsecond insulating substrate, and wherein said auxiliary sample supplyport is disposed between said air hole and said sample supply port. 26.A biosensor as defined in claim 13, wherein said auxiliary sample supplyport is formed in one of said insulating substrates, said sample supplyport is formed between said insulating substrates, and said air hole isformed in one of said insulating substrates.
 27. A biosensor as definedin claim 13, further comprising a reagent layer disposed between thespacer and the second insulating substrate, the reagent layer being influid communication with the capillary.